You are right, the status and future of AAA has been discussed a few times
in this forum, with the overall observations that a) not many
instrument/reagent companies are devoting resources to develop or even
support AAA technology, b) not many labs are doing it routinely anymore
because it can be costly to maintain; c) those labs that still do AAA have
to use increasingly-aging instruments (and, on a personal note,
increasingly-aging analysts!), d) since it isn't a 'glamorous' analytical
technique and/or because fewer labs are still supporting it, not many
early-career scientists are getting experienced in the finesse of the
methodology. Nonetheless, for the characterization of proteins and peptides
used in biotechnology products it still is a key method and is required in
most product submissions as a release method for compositional analysis.
I would not say that the data are not accurate enough for today's standards,
especially with improvements in HPLC and data analysis technology. The
greatest variability comes from hydrolysis steps, and inter-lab testing
(which might be expected given differences in analyst's experience).
Because of the importance of control of hydrolysis conditions for reliable
results, I was quite dismayed when I learned that Waters was discontinuing
the sale of their PicoTag Workstations. I understand that the sales volume
for these had dropped off such that it was no longer feasible to carry them
:-( One set of discussions involved some creative suggestions from our
experienced ABRF listeners on how to make alternate hydrolysis systems.
Unfortunately, many of these would not be suitable for GLP/cGMP labs where
instrument validation is a requirement.
I have worked with ABI 421s using PTC chemistry, and now work with HP
AminoQuant and (soon) with Waters AcuTaq chemistry. There were several
mechanical problems in using the 421s in a high-throughput cGMP setting with
a wide variety of proteins and variable experience level of analysts,
causing numerous instrument service calls. But the PTC chemistry was very
good. In my current lab we use AminoQuant with a diversity of proteins and
peptides, and we run GLP/cGMP analyses nearly every day. I am very pleased
with the performance of the chemistry and of the instrument. In the next
month or so we will add AcuTaq AAA as a second method, using an Alliance
HPLC system. I will be interested to see how the data look between the two
methods, especially since they will use the same hydrolysis systems and same
analysts. I have never used the Beckman system, although I have worked with
data generated from such a system in another lab. The data looked very good,
but the analyst was a very experienced protein chemist who was known for
excellent work, so I can't tell you how much was art and how much science. I
recently became aware of a new approach being taken by Dionex for
non-derivatized AAA using HPAE-PAD technology. As yet I haven't seen the
methods or any data, but it is intriguing.
Some of us in the ABRF have debated the value of continuing to conduct
proficiency testing via the ABRF AAA committee. My position has been that
we need such inter-lab and multi-method comparisons now as much as ever.
The objective of these studies was to establish the performance expectations
of the technology. I still see that need, especially as the existing
instruments age. When someone wants to know the real state of the art in
AAA, they might be misled on performance capabilities from a 10 yr old study
if such performance is no longer possible to obtain. While these studies
might be done every 2-3 years rather than annually, they still would yield
valuable information about amino acid analysis. Given the number of core
labs discontinuing AAA, I expect that our n number would be considerable
smaller than before, unless new ABRF members from biotech labs where AAA
might still be used extensively would participate. I'd be curious to add
the question of how many of these labs are using validated AAA methods to
see if this affects the accuracy and precision of their data. Perhaps if
new AAA technologies (and more committee volunteers?) do spring up, the
interest in interlab method comparisons will grow.
Nadine Ritter, Ph.D.
Director
Analytical Services
BioReliance
Weather inconsequential: The heat wave has finally broken in D.C. - but the
drought continues. My cherry tomatoes and herb garden have been a
substantial source of nourishment for the birds and bunnies, leaving little
for the humans. Oh well...it is a small price to pay to stay in harmony
with nature, I suppose.
_____________________
Dear Sam Massoni,
It is not you, the industry has seemed to disappeared from my
perspective as well. It may be that the rigors of the analysis are just to
great and the data just is not accurate enough for today's standards? To be
honest I don't really know.
You asked for the latest and greatest and I have seen some of the
responses posted on this bulletin board. Both Hitachi and Dionex are new for
me and I would be very interested in gaining perspective on these
instruments. Personally, I have experience using both PE/ABI 421, PITC
precolumn derivitization and Beckman's strange kind of slap dashed system,
post column ninhydrin. I have ordered Millipore's product for pre column
derivitzation and will being testing it's application using a H/P 1100
system. I need to keep whatever methods I use within the cGL/MPs guidelines
and make sure that what ever system I use is validatable and transferable as
well. Please feel free to pursue this line of communication off or online.
James G. Freedy
Scientist
OrthoBiotech, Inc.
Raritan, N.J.
jfreedy@obius.jnj.com
(908) 218-7295
----------
From: New England Peptide, Inc.[SMTP:nep@net1plus.com]
Sent: Tuesday, June 29, 1999 5:22 PM
To: Recipients of ABRF List
Subject: AAA instruments
HI ABRF'ers
Does anyone know who has the latest and greatest instruments for
doing amino acid analysis. Is it me or has the industry disappeared. Any
suggestions would be great on which new models are decent.
Thanks
Sam Massoni
New England Peptide, Inc.
Nadine Ritter, Ph.D.
Director, Analytical Services
BioReliance Corp.
301-610-2587